Oct 2011 SDMBT Lecture 9 Mass Spectrometry. Oct 2011 SDMBT Objectives Describe the principle of...
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Transcript of Oct 2011 SDMBT Lecture 9 Mass Spectrometry. Oct 2011 SDMBT Objectives Describe the principle of...
Oct 2011 SDMBT
Lecture 9Mass Spectrometry
Oct 2011 SDMBT
Objectives
Describe the principle of Mass Spectrometry
In particular, the ionisation methods(a) MALDI(b) ESI
Oct 2011 SDMBT
General workflow for proteomic analysis
SampleSample preparation
Protein mixture Sample separation and visualisation
Comparative analysis
DigestionPeptides
Mass spectrometry
MS dataDatabase search
Protein identification
Oct 2011 SDMBT
Protein Identification
MALDI-TOF
Molecular weight of tryptic peptides
ESI-MS
Molecular weight of protein
MS/MS
Molecular weight of peptide fragments
Oct 2011 SDMBT
Mass spectrometry workflowTypical process
(Medical College of Georgia)
ionisation
acceleration
sorting
detection
Mass-to-charge ratio
(m/z)
Oct 2011 SDMBT
The need for ionisation
Analyte has to be converted into gas-phase ions – only charged ions can be detected by MS
Movement of gas-phase ions can be precisely controlled by electromagnetic fields
Difficulty of generating gas-phase ions results in complex instruments and high costs – need high vacuum
Gas phase ions can be generated by
e.g. an electron beam (electron ionisation)
collision with inert gas molecules (MS-MS)
a laser beam (MALDI-TOF)
a charged nozzle (ESI-MS)
Oct 2011 SDMBT
A plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly corresponding to the number of ions) (y-axis)
Mass spectrum of water – ionised by electron ionisationNotice that the water molecule fragments upon ionisation
Mass Spectrum
H2O+
HO+
O+
H+
Oct 2011 SDMBT
HCHO+
CHO+
CO+
HCHO2+
HCH+
CH+
C+
Notice: the horizontal axis is mass/charge ratio of ions (m/z)
Oct 2011 SDMBT
The need for ionisation
Proteins are large macromolecules – electron ionisation works only for volatile compounds – also electron ionisation is a ‘hard’ ionisation technique – molecule fragments into smaller ions
Need to find an efficient method to convert proteins from liquid phase to gas-phase ions
Soft ionisation methods like electrospray ionisation (ESI) and matrix-assisted laser-desorption ionisation (MALDI)
Oct 2011 SDMBT
Soft ionisationIonise peptides into gas-phase ions
Too much energy (hard ionisation) breaks peptides into smaller fragments
(Prof. Jose-Luis Jimenez)
Oct 2011 SDMBT
Maldi TOF/TOF mass spectroscopic spectre for UCH-L3 digested Ecotin-Ubiquitin-FLS in the m/z range 799–4013 . The peaks
at 2683 and 1342 represent the single and double protonation states of the FLS peptide, respectively.
MALDI-TOF mass spectrumPeptide does not fragment
Soft ionisation
Oct 2011 SDMBT
MALDI
Peptides are mixed with a matrix, which crystallise on a metal plate
Peptides protonated by matrix and solvent
Pulses of laser transfer energy to matrix
Matrix vaporises with peptide samples
Oct 2011 SDMBT
MALDI
Oct 2011 SDMBT
MALDI plate
(Bruker Daltonics)
(University of Pittsburgh BRSF)
Oct 2011 SDMBT
MatrixContains ring structures to absorb energy from UV laser
Contains acid group to protonate peptides
OH
OH
HOOC
2,5-dihydroxybenzoic acid
OH
COOH
CN
-cyano-4-hydroxycinnamic acid
Matrix solution – DHB or CHCA dissolved in acetonitrile and trifluoroacetic acid
Sinapinic acid
Oct 2011 SDMBT
Characteristics of MALDI
Efficient protonation of peptides
Ions generated in discrete packets due to laser pulses
Combined with time-of-flight (TOF) mass analysis to give very high sensitivity
More tolerant than ESI to contaminants (urea, SDS), thus HPLC separation is not required
MALDI-TOF of mixture of cytochrome, ubiquitin, myoglobin(without PSD) – note single peaks (except cytochrome) – cantell Mw of 3 proteins in one spectrum
Mw of myoglobin17000
Mw of cytochrome C 12000
Mw of ubiqutin 8500
Oct 2011 SDMBT
Oct 2011 SDMBT
Trypsin digested peptides
(ExPasy PeptideCutter)
Arginine or Lysine Proline
From last lecture: one spot in 2D-gel represents one protein – this proteinis digested with trypsin into smaller peptides.The peptide mixture is spotted on the MALDI plate
Oct 2011 SDMBT
King’s College London
(Pierce)
Major peaks at:
646 742 748
780 830 2186
Represent expected sizes of tryptic peptides
Soft ionisation ensures no further fragmentation of peptides
MALDI spectrum of Tryptic digest of β-casein
Each peak represents one peptidedigested by trypsin from casein
Oct 2011 SDMBT
Tryptic digest of β-casein
King’s College London
(Pierce)
Most abundant peak base peak, abundance set to 100%
MS is seldom quantitative, only tells relative abundance
Ideally every peptide is ionised to same extent 100%
Oct 2011 SDMBT
Electrospray Ionisation (ESI)Peptides in acidic solution are pushed out of a fine capillary
A high positive charge at the capillary results in the formation of a Taylor cone
Peptides protonated in charged droplets and ionised
Gas-phase ions repelled from capillary into instrument
Nitrogen aids in evaporation of solvent from charged droplets
Oct 2011 SDMBT (Royal Society of Chemistry)
Electrospray Ionisation (ESI)
Oct 2011 SDMBT (New Objective, Inc)
High voltage results in accumulation of positive charges on droplets
Increased charge density results in instability of droplets
Droplets break down successively into smaller sizes
Electrospray Ionisation (ESI)
Oct 2011 SDMBT
Characteristics of ESI
Acidic conditions result in protonation of all basic sites in peptides
Basic side groups of lysine, arginine, histidine
Produces peptide ions that are multiply protonated
Advantageous for peptides digested by trypsin as doubly charged peptides are formed
Oct 2011 SDMBT
Characteristics of ESI
Efficient ionisation process results in sensitivity of ESI experiments
General compatibility of ESI with reversed-phase high performance liquid chromatography (RP-HPLC) LC/MS-MS
ESI-MS - often see doubly, triply, multiply charged ions
Oct 2011 SDMBT
ESI of myoglobin
Mw=1413.8x12-12 = 16953.6
Mw – need to know how to interpret by assigning charges
Compare with slide 17More complicated Different scale on horizontal axis
Oct 2011 SDMBT
Oct 2011 SDMBT
ESI of cytochrome C
Compare with slide 17More complicated Different scale on horizontal axis
ESI of ubiquitin
5+
Mw = 8484.5
6+
7+
Compare with slide 17More complicated Different scale on horizontal axis
Oct 2011 SDMBT
MALDI-TOF – 3 peaksEach of each protein
ESI – complicatedif sample was a mixture – 3 superimposedspectra
Oct 2011 SDMBT
Oct 2011 SDMBT
MALDI-TOF ESI
Sample Solution but ends upembedded in crystalline matrix
Sample tolerant to salts
Singly charged ions
Adduct-formation not so
Common.
Appearance – one peak
Solutioneg can come straight from HPLC
Results can be affected by salts eg phosphates
Multiply-charged ions
Adducts with salts common
May be difficult to interpret spectra
Appearance – many peaks
due to multiple charges
Comparison of ionisation methods
Oct 2011 SDMBT
MALDI-TOF ESI
Ionisation Laser Charged spray nozzle
Mass analyser TOF Quad
Fragmentation is made possible by
PSD (Post-Source decay)
“Pseudo-MS-MS”
MS-MS or Tandem MS
Ions fragmented by collision with inert gas
Use Mainly peptides Peptides and proteins
Comparison of ionisation methods
Oct 2011 SDMBT
Vacuum SystemVacuum System
Sample Sample InletInlet DetectorDetector
Data Data SystemSystem
Mass Mass AnalyserAnalyser
Ionisation Ionisation MethodMethod
Components of a mass spectrometer
Atmosphere
(adapted from Thermo Finnigan)
MALDI
ESI
TOF
Quadrupole
Ion trap
Combination
ionisation acceleration detection
Oct 2011 SDMBT
The need for a mass analyser
Gas-phase ions has to be filtered/arranged in order to allow selection of specific ions for further analysis.
Movement of gas-phase ions precisely controlled by use of electromagnetic fields in a mass analyser
Main types of mass analyser-TOF (time-of-flight analyser)
-Quadrupole analyser
-Fourier-Transform Ion Cyclotron Resonance
-Orbitrap
Oct 2011 SDMBT
TOF
Time-of-flight (TOF) analyser is the simplest mass analyser
Peptide ions accelerated into flight-tube, and maintains a velocity due its given kinetic energy
TOF analyzer requires that ions are introduced in a pulse well-suited for MALDI
Resolution increases with length of TOF tube
Oct 2011 SDMBT
Linear TOF
(John Lennon, University of Washington)
Oct 2011 SDMBT
MALDI- TOF with reflectron
Reflectron - This turns ions around in an electric field, sending
them towards the detector – improves mass resolution
Oct 2011 SDMBT
Quadrupole
(NASA)
Made up of 4 parallel gold barsComplex electromagnetic fieldset up which allows only ions of aa set mass/charge ratio through
Oct 2011 SDMBT
Flight path of an ion through a quadrupole
Correct m/z ratio Larger/smaller m/z ratio
Ions that spiral out of control crash into the rods or casing
Oct 2011 SDMBT
Fourier Transform Ion cyclotron resonance (FT-ICR)
See this websitehttp://www.chm.bris.ac.uk/ms/theory/fticr-massspec.html
Magnetic fields applied to trapping plates constrain the ions to move around in a circlein between the plates. The circular motion induces an alternating current.The frequency of the AC is related to the m/z ratio.
Main advantage of FT-ICRis very high mass resolution
Oct 2011 SDMBT
Orbitrap
Image from thermo.com
Similar to FT-ICRElectric field applied to trapping plates constrain the ions to move around in a circlein between the plates. The circular motion induces an alternating current.The frequency of the AC is related to the m/z ratio.
Inner electrode
Outer electrode
Main advantage of Orbitrapis very high mass resolution
Oct 2011 SDMBT
Tandem MS (MS/MS)
MALDI and ESI are soft ionisation techniques – peptides or proteinsdo not fragment – useful for molecular weight determination
However sometimes fragmentation is useful because it give useful informationabout the amino acid sequence of peptides (next lecture)
Fragmentation can achieved by a number of ways
-Post source decay (PSD)-Collision induced dissociation (CID)-Infrared multiphoton dissociation-Electron capture dissociation (ECD)-Electron transfer dissociation (ETD)
Oct 2011 SDMBT
Post-Source Decay (PSD)
- Use higher laser power or introduce inert gas to collide with ions so that ions fragments between source and TOF
- Method of fragmenting intact peptides - get amino acid sequence information (see next lecture)
- Electronics to select ions to go into the TOF
http://abrf.org/ABRFNews/1995/December1995/dec95maldi.html
Oct 2011 SDMBT
(K. Yoshinari, Rapid Commun. Mass Spectrom. 14, 215-223, 2000)
Ion trap
(NASA)
All other dissociation techniques involve the use of an ion trapIon trap essentially a quadrupole where the magnetic field is set such that a particular ion is trapped in the space between the electrodes
Oct 2011 SDMBT
Ion trap
(March, JMS Vol. 32, 351-369, 1997 )
-Collision induced dissociation (CID) – inert gas (e.g. Xe, Ar, N2 or He) is introduced into trap, collisions will cause peptides to fragment usually the C-N peptide bond to produce b and y ions (see later)-Infrared multiphoton dissociation (IRMPD) – IR laser is fired into ions to excite the vibrations of peptides. -Electron capture dissociation (ECD) – electron beam is fired into ions to produce c and z ions-Electron transfer dissociation (ETD) – singly charged anion, fluoranthracene is introduced. An electron is transferred to peptide. Results in c and z fragments
Oct 2011 SDMBT
Hybrid mass analysers
Basically a combination of two types of mass analysers or many of the same type
Each analyser performs a different function
Used to perform tandem mass spectrometry, using collision induced dissociation (CID)
Oct 2011 SDMBT
Tandem quadrupoles (QQQ/TSQ)
Video
Quantitative Chemical Analysis
Select specific parent ion
CID with inert gas
Scanning of all m/z of daughter ions
Oct 2011 SDMBT
Tandem mass spectrometry
A) MS spectrum of HSP27. The peptide whose MS/MS spectrum is shown in panel B is indicated. B) MS/MS spectrum of the peptide ion m/z 1163 obtained in CID mode.Perroud et al. Molecular Cancer 2006 5:64